Antifungal activity of metabolites of the endophytic fungus Trichoderma

brevicompactum from garlic

Xuping Shentu1,2, Xiaohuan Zhan2, Zheng Ma2, Xiaoping Yu2, Chuanxi Zhang1

1Institute of Insect Science, Zhejiang University, Hangzhou, China.2College of Life Science, China Jiliang University,

Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, Hangzhou, China.

Submitted: June 25, 2012; Approved: April 1, 2013.

Abstract

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma

brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8S-

ITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by

bioactivity-guided fractionation and identified as 4�-acetoxy-12,13- epoxy-�9-trichothecene (tricho-

dermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity

on Rhizoctonia solani, with an EC50 of 0.25 �gmL-1. Strong inhibition by trichodermin was also

found for Botrytis cinerea, with an EC50 of 2.02 �gmL-1. However, a relatively poor inhibitory effect

was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 �gmL-1).

Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity

on the above phytopathogens. There is little known about endophytes from garlic. This paper studied

in detail the identification of endophytic T. brevicompactum from garlic and the characterization of

its active metabolite trichodermin.

Key words: Trichoderma brevicompactum, garlic, endophytic fungus, Trichodermin, antifungal ac-

tivity.

Introduction

An increased concern over the widespread use of

toxic agricultural chemicals and an increased recognition of

the potential of biological pesticides has stimulated much

research to develop and implement the use of biological

pesticides (Epstein and Bassein, 2003; Strobel and Daisy,

2003). Chemical pesticides have been the objects of sub-

stantial criticism in recent years, mainly due to their ad-

verse effects on the environment, human health and other

non-target organisms (Raju et al., 2003). The potential use

of microbe-based biocontrol agents as replacements or sup-

plements for agrochemicals has been addressed in many re-

cent reports (Hynes and Boyetchko, 2006; Vasudevan et

al., 2002). Endophytes are microorganisms that live in the

intercellular spaces of stems, petioles, roots and leaves of

plants, and there is no discernible manifestation of their

presence (Strobel and Long, 1998; Yang et al., 2011). The

symbiosis between plants and endophytes is well known;

specifically, the former protects and feeds the latter, which

‘in return’ produces bioactive (plant growth regulatory, an-

tibacterial, antifungal, antiviral, insecticidal, etc.) sub-

stances to enhance the growth and competitiveness of the

host in nature (Carroll 1988; Konig et al., 1999; Yang et al.,

1994). Accordingly, endophytes are currently considered to

be a wellspring of bioactive secondary metabolites with the

potential for medical, agricultural, and/or industrial exploi-

tation (Schulz et al., 2002; Tan et al., 2000).

Garlic (Allium sativum), a member of the family

Liliaceae, contains an abundance of chemical compounds

that have antimicrobial, anticancer and antioxidant activity

(Omar and Al-Wabel, 2010). However, little is known

about the secondary metabolites of endophytes harbored

inside the healthy garlic tissues. To find fungi for poten-

tially efficient biopesticides, we isolated the endophytic

Brazilian Journal of Microbiology 45, 1, 248-254 (2014) Copyright © 2014, Sociedade Brasileira de Microbiologia

ISSN 1678-4405 www.sbmicrobiologia.org.br

Send correspondence to X. Yu. College of Life Sciences, China Jiliang University, 310018 Hangzhou Zhejiang Province, P.R. China. E-mail:

yuxiaoping19630306@163.com.

Research Paper

isolates from plants such as garlic and carried out a bio-

assay against phytopathogens. The candidate isolate 0248

showed strong inhibitory activity against phytopathogens.

In this paper, the identification of the endophytic fungus

isolate 0248 from garlic by morphological and molecular

characteristics is described. The purification and character-

ization of antifungal metabolites from the fermentation

broth of isolate 0248 are also studied.

Materials and Methods

Endophytic fungus and phytopathogenic strains

The endophytic fungus 0248 was isolated from

healthy garlic based on antifungal activity. The phyto-

pathogenic fungal strains, including Fusarium oxysporum,

Colletotrichum lindemuthianum, C. ampelinum,

Rhizoctonia solani and Botrytis cinerea, were kindly pro-

vided by the Institute of Plant Protection and Microorgan-

isms, Zhejiang Academy of Agricultural Sciences. These

strains were separately stored on PDA slants containing po-

tato-dextrose agar at 4 °C until used.

Culture conditions for isolate 0248

Fresh mycelium were picked and inoculated into 500

mL Erlenmeyer flasks containing 100 mL PD medium. Af-

ter 2 days of incubation at 28 � 1 °C on a rotary shaker at

150 rpm, 3 mL culture liquid was transferred as seeds into a

300-mL Erlenmeyer flask containing 30 mL medium

(20 gL-1 dextrose, 5 gL-1 peptone, 1 gL-1 beef extract,

0.001 gL-1 ZnSO47H2O and 0.01 gL-1 NH4Cl). The result-

ing culture was kept on a rotary shaker at 180 rpm for 4 days

at 28 � 1 °C. The fermentation broth was separated from the

mycelia by filtration through filter paper using a Buchner

funnel. One hundred milligrams mycelial biomass was

taken following washing (two times) with sterile Tris-

EDTA buffer for DNA extraction.

Identification of the endophytic fungus isolate 0248

The candidate isolate 0248 was identified by using

morphological and molecular procedures. The morphologi-

cal examination was performed by observing the character-

istics of fungal culture on PDA, yeast extract sucrose agar

(YES)(40 gL-1yeast extract, 160 gL-1 sucrose and 20 gL-1

agar) and synthetic low-nutrient agar (SNA) (1.0 gL-1

KH2PO4, 1.0 gL-1 KNO3, 0.5 gL-1 MgSO47H2O, 0.5 gL-1

KCl, 0.2 gL-1dextrose, 0.2 gL-1 Sucrose and 20 gL-1 agar) in

Petri dishes, separately. Microscopic slides were prepared

and examined under a light microscope (Leica, German).

Total genomic DNA was extracted from fresh mycelium

according to the cetyltrimethylammonium bromide

(CTAB, Sigma) procedure (Cappiccino and Sherman,

1996).

A region of nuclear rDNA, containing the internal

transcribed spacer regions 1 and 2 and the 5.8S rDNA gene

region, was amplified by polymerase chain reaction (PCR)

using the primer pair ITS1

(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4

(5’-TCCTCCGCTTA TTGATATGC-3’) with the follow-

ing protocol: 1 min initial denaturation at 94 °C, followed

by 30 cycles of 1 min denaturation at 94 °C, 1 min primer

annealing at 50 °C and 90 s extension at 74 °C, and a final

extension period of 7 min at 74 °C. A fragment of tef1 was

amplified by the primer pair tef1 fw

(5’-GTGAGCGTGGTATCACCATCG-3’) and tef1 rev

(5’-GCCATCCTTGGAGACCAGC-3’) with the follow-

ing amplification protocol: 1 min initial denaturation at

94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at

59 °C and 50 s at 74 °C, and a final extension period of

7 min at 74 °C (Kraus et al., 2004). PCR products were se-

quenced by Shanghai Invitrogen Co. (Shanghai, China).

The DNA sequence obtained was submitted to GenBank

and TrichoKEY for homology analysis by BLASTN and

TrichoKEY 2 program, respectively. Phylogenetic trees

were inferred using the neighbor-joining (NJ) algorithm

(Saitou and Nei, 1987) from the PHYLIP suite programs

(Felsenstein 1995). The topologies of the resulting

unrooted trees were evaluated in a bootstrap analysis

(Felsenstein 1985) based on 500 resamplings of the NJ

dataset using the PHYLIP package.

Analytical conditions

Silica gel (200-300 mesh) for column chromatogra-

phy and GF254 (30-40 �m) for TLC were produced by

Qingdao Marine Chemical Factory, Qingdao, China. All

other chemicals used in this study were of analytical grade.

IR spectra were recorded in KBr disks on an FTIR-8400S

instrument. All NMR experiments were performed on a

Bruker AM 500 FTNMR spectrometer using TMS as the

internal standard. Mass spectrometry (MS) data were ob-

tained using an Agilent-5873 mass spectrometer at 70 eV.

Antifungal assay

Antifungal activity was tested with the hyphae

growth velocity assay (Lu et al., 2000). The effective con-

centration of the sample causing a 50% inhibition of my-

celial growth (EC50) was determined. The commercial anti-

fungal agent carbendazim, produced by NingXia Sanxi

Chemical Co., Ltd. China, was used as positive control.

Mycelial discs (4 mm in diameter) of phytopathogenic

fungi grown on PDA were cut from the margins of the col-

ony and placed on the same medium containing different

concentrations of the sample. A negative control was main-

tained with sterile water mixed with PDA medium. Each

treatment had three replicates. The diameter of colony

growth was measured when the fungal growth in the con-

trol had completely covered the Petri dishes. The inhibition

percentage of mycelial growth was calculated as follows:

Mycelial growth inhibition (%) ��

�D D

D

a b

a

100

249 Shentu et al.

where Da represents control colony diameter and Db repre-

sents treated colony diameter. The colony diameter is in

millimeters.

Gas chromatographic analysis

The active compound was dissolved in HPLC grade

methanol and quantified by GC (Agilent 6890N) with an

HP-5 capillary column (5% phenyl methyl siloxane, 30 m x

320 �m x 0.25 �m) with an FID detector and nitrogen as the

carrier gas. The injector temperature was 250 °C, the detec-

tor temperature was 270 °C, the column temperature rose

by program with an initial temperature of 100 °C main-

tained for 5 min and rising to 260 °C at a rate of 10 °C min-1

and maintained for 5 min. The flow rate of N2 was

1.5 mL min-1.

Statistical analysis

All statistical analysis were performed using SPSS

15.0 software. The log dose-response curves allowed deter-

mination of the EC50 for the fungi bioassay according to

probit analysis. The 95% confidence limits for the range of

EC50 values were determined by the least-square regression

analysis of the relative growth rate (% control) against the

logarithm of thecompound concentration(Rabea et al.,

2009).

Results

Identification of the endophytic fungus isolate 0248

On PDA medium, the isolate 0248 mycelium grew

rapidly at 28 � 1 °C in darkness. The aerial mycelia were

white to green and colorless on the reverse side of the plate.

After 6 days at 28 � 1 °C conspicuous concentric rings were

present, and colonies with a regular margin were 90 mm in

diameter. No diffuse pigment or odor was noted. However,

cultures of isolate 0248 grown on YES and SNA tended to

form pustules and grew slowly. Colonies did not grow radi-

ally on SNA. This result might be due to component differ-

ence of PDA, SNA and and YES. All colonies sporulated

on PDA, SNA and YES medium at 28 � 1 °C in the dark.

On PDA, conidiomata grew from stromata and consisted of

single conidiophores that were grey-green, smooth, and

oval to spherical. The diameter of conidiophores was

5.5-7.0 �m (Figure 1).

The sequences of the region of ITS1-5.8S- ITS2

(GenBank accession number JN257218) and the fragment

of tef1 (GenBank accession number JN257219) were ob-

tained in this study. The comparison of ITS1-5.8S- ITS2

and tef1 genes of isolate 0248 with sequences in the

GenBank database revealed that this endophytic fungus

was highly homologous to different strains of Trichoderma

brevicompactum, with nucleotide identities 99-100% for

ITS1-5.8S- ITS2 and 97-100% for tef1. The topology of the

phylogram in Figure 2 confirmed that 100% bootstrap val-

ues were detected between the ITS1-5.8S- ITS2 and tef1 se-

quences of isolate 0248 and those of T. brevicompactum.

Therefore, isolate 0248 was closely related to T.

brevicompactum.

TrichoKEY2 is a program for the quick molecular

identification of Hypocrea and Trichoderma on the genus

and species levels based on an oligonucleotide DNA

BarCode (www.isth.info), a diagnostic combination of sev-

eral oligonucleotides (hallmarks) specifically allocated

within the internal transcribed spacer 1 and 2 (ITS1 and 2)

sequences of the rRNA gene cluster (Druzhinina et al.,

2005). Identification of isolate 0248 using the

oligonucleotide barcode showed that it belongs to the spe-

cies T. brevicompactum and that the identification reliabil-

ity was high.

Extraction and purification of metabolites of theendophytic fungus

The isolation of active compounds from strain 0248

was carried out with a bioassay against R. solani, and the

fractions with antifungal activity were used for further iso-

lation. The fermentation broth (total volume 30 L) of isolate

0248 was collected and extracted three times with ethyl ac-

etate (filtrate/ethyl acetate 1:1, v/v) at ambient temperature.

A brown residue (8.25 g) was obtained by evaporating the

solvent from the extract in a vacuum; the residue was sepa-

rated by chromatography over a silica gel column (200 g,

100-200 mesh), eluting successively with 1000 mL each of

petroleum ether/ethyl acetate (v/v, 30:1, 20:1, 10:1, 8:1,

6:1, 4:1, 2:1, and 1:1) and acetone to obtain 8 fractions

(A1~A8). A5 was re-chromatographed over a silica gel col-

umn (50 g, 100-200 mesh) eluted with 500 mL each of pe-

troleum ether/ethyl acetate (v/v, 20:1, 15:1, 10:1, 5:1, and

1:1), ethyl acetate and acetone, obtaining 4 fractions

(4B1~4B4). Based on the TLC monitoring and antifungal

test, 4B2 was further separated over a silica gel column

(25 g, 100-200 mesh) eluted with 100 mL each of petro-

leum ether/ethyl acetate (v/v, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,

4:1, 3:1, 2:1 and 1:1) and acetone to give fractions

5C1~5C15. 5C9 and 5C10 were combined to yield 258 mg

active compound T2.

Structure elucidation of antifungal compound T2

Compound T2, which had strong antifungal activity,

was identified as 4�-acetoxy-12, 13-epoxy-�9-trichothe-

cene (trichodermin) by spectral analysis, MS data and di-

rect comparison with published information (Godtfredsen

and Vangedal, 1965). Colorless crystal (pentane), mp

45-46 °C; + 10.2° (c 0. 1 mg mL-1, CHCl3); IR: [ ] D

20 (cm-1)

2900, 1732, 1374, 1243 and 1080 cm-1; The molecular for-

mula of compound T2 was determined to be C17H24O4 by its

spectral data (1H, 13C NMR and DEPT), MW 292.16746,1H NMR (CDCl3, 400 MHz) : 5.506(dd, 1H, J = 6.4,

3.2 Hz, H-4), 5.337-5.350(m, 1H, H-10), 3.757(d, J = 4.0

Hz, 1 H, H-2), 3.546(d, 1H, J = 4.4 Hz, H-11), 3.059(d, 1 H,

Endophytic Trichoderma from garlic 250

J = 3.2 Hz, H-13), 2.768 (d, J = 3.2 Hz, 1H, H-13’),

2.473(dd, J = 12.4, 6.4 Hz, 1H, H-3), 2.021(s, 3H, H-18),

1.951~1.865(m, 4 H, H-8, H-8’, H-7, H-3’), 1.653 (s, 3H,

H-16), 1.354(m, 1H, H-7’), 0.875(s, 3H, H-15), 0.658 (s ,

3H, H-14). 13C NMR (CDCl3, 500 MHz) : 170.71(C-17),

139.94(C-9), 118.42(C-10), 78.89(C-2), 74.86(C-4),

70.28(C-11), 65.30(C-12), 48.70(C-5), 47.62(C-13),

40.19(C-6), 36.44(C-3), 27.77(C-8), 24.24 (C-7),

23.06(C-16), 20.93(C-18), 15.78(C-15), 5.60(C-14). The

spectrum of trichodermin used for quantitative analysis by

GC is shown in Figure 3.

Antifungal activities

The results of the antifungal activity of trichodermin

against R. solani, F. oxysporum, C. lindemuthianum, C.

ampelinum and B. cinerea were expressed as EC50 and are

presented in Table 1. We observed that trichodermin mark-

edly inhibits R. solani, with an EC50 of 0.25 �gmL-1.

Trichodermin also strongly inhibits B. cinerea, with an

EC50 of 2.02 �gmL-1. However, trichodermin had a poor in-

hibitory effect against C. lindemuthianum (EC50 = 25.60

�gmL-1). The results also indicated that the inhibitory effect

of trichodermin differed with regard to the type of the fungi.

251 Shentu et al.

Figure 1 - Morphology of isolate 0248. A, Appearance of colonies on YES; B, Appearance of colonies on PDA; C, Appearance of colonies on SNA; D,

Appearance of pustules on SNA (arrow); E, Appearance of pustules on YES (arrow); F-I, hypha and conidia.

Compared with the positive controls, trichodermin has

broad-spectrum and strong antifungal activity.

Discussion

In our efforts to screen for suitable microorganisms

from garlic that can produce bioactive compounds, we

found that isolate 0248, subsequently identified as T.

brevicompactum, showed strong antifungal activities. This

is the first study of the endophytic fungus from garlic. T.

brevicompactum, a new species, was first confirmed by

morphological, molecular, and phylogenetic analyses in

2004, and related research has been very rare. Trichoderma

species are common soil-borne fungi. One of the most sig-

nificant ecological niches occupied by Trichoderma spe-

cies is the plant rhizosphere (Harman et al., 2004). The

concept of Trichoderma as an endophyte has received a less

attention.

Although Trichoderma isolates were isolated from

live sapwood below the bark of trunks of wild and culti-

vated Theobroma cacao and other Theobroma species (Ev-

ans et al., 2003), only a small number of Trichoderma

Endophytic Trichoderma from garlic 252

Table 1 - Antifungal activities of trichodermin on mycelial growth of

pathogenic fungi.

Microorganisms EC50 (�gmL-1)

Trichodermin Carbendazim

Rhizoctonia solani 0.25 0.36

Fusarium oxysporum 8.51 10.35

Colletotrichum lindemuthianum 25.60 > 50.00

Colletotrichum ampelinum 16.65 > 50.00

Botrytis cinerea 2.02 > 50.00

Figure 2 - A Neighbor-joining tree based on ITS rDNA sequence of isolate 0248 and its closest ITS rDNA matches in GenBank. Values on the nodes indi-

cate bootstrap percent confidence. B Neighbor-joining tree based on tef1 sequence of isolate 0248 and its closest tef1 matches in GenBank. Values on the

nodes indicate bootstrap percent confidence.

Figure 3 - Representative gas chromatography of trichodermin isolated

and purified from fermentation broth by isolate 0248.

isolates from the collection have been studied for their

biocontrol activities (Bailey et al., 2008; Holmes et al.,

2004; Samuels et al., 2006a, 2006b). However, it is unclear

which compounds produced by these isolates have inhibi-

tory activity against phytopathogens (Howell 2003). Purifi-

cation of secondary metabolites from fermentation broths

can be a challenging task due to the complexity of the me-

dium, the inherent instability of the molecular structures or

by the action of enzymes present in the fermentation broth,

leading to poor isolation yield and loss of antibiotic activ-

ity. In this paper, we studied the isolation and purification

of an active metabolite from the endophytic T.

brevicompactum fermentation broth in detail and identified

trichodermin as the main active compound.

It is well known that trichodermin is a very potent in-

hibitor of protein synthesis (Carrasco et al., 1973). Reports

of trichodermin activity against phytopathogens are less

common. We studied the inhibitory activity of trichoder-

min against mycelial growth of plant pathogenic fungi and

showed that it has potential value in agricultural applica-

tions. It was found that endophytic T. brevicompactum

from garlic produced approximately 86 mgL-1 trichodermin

in liquid cultures. This is a marked increase in trichodermin

production compared with previous reports, in which T.

harzianum Th008 produced approximately 92.67 �gL-1

trichodermin in liquid cultures of PDB (Bertagnolli et al.,

1998). Therefore, the high production of trichodermin and

its high antifungal activity highlight the potential of this

endophytic T. brevicompactum isolate in antibiotic produc-

tion. In addition, very little was previously known about the

nature of the relationship between T. brevicompactum and

the garlic plant. Whether trichodermin has any influence on

garlic growth is also an interesting possibility and should be

researched in future.

Acknowledgments

This work was supported by National Natural Science

Fundation of China (30940018), Zhejiang Province Pro-

grams for Science and Technology Development

(2010C32063, 2011R09027-03, and 2013C32008) and

Zhejiang Province Natural Science Fundation

(LY12C14012)

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